All cells must faithfully replicate their genomes in order to reproduce. Unrepaired damage that blocks replication can lead to a loss of genomic stability, mutations, or cell death. Despite the widely accepted view that most genomic instability and mutagenesis results from the replication of damaged DNA, the cellular mechanism(s) by which replication blocking lesions are processed and repaired remains largely uncharacterized. The RecF protein of E.coli belongs to a ubiquitous group of recombination mediators (RMs) that include eukaryotic proteins such as Rad52 and BRCA2 and are required to maintain genome stability in the presence of DNA damage. Several human homologs of RecF pathway proteins- BLM, WRN, Rothmund-Thompson syndrome, are associated with genome instability, premature aging, and cancer when mutated. These processes have proven extremely difficult to approach using biochemical or cellular approaches in humans, making E. coli an extremely attractive and appropriate model system for understanding the molecular basis of these complex diseases. This project seeks to identify the cellular mechanism by which RecF initiates the processing and repair of arrested replication forks. We have previously shown that RecF is essential to maintain the integrity of DNA ends at replication forks disrupted by DNA damage and have recently obtained the crystal structure of the protein. Using cellular assays previously established in our lab, we will characterize RecF varients with altered functions that will allow us to differentiate between the alternative hypotheses that i) RecF functions as a clamp loader to recruit RecA and other processing enzymes to the arrested replication fork, ii) RecF functions with RecA as a motor protein that catalyzes the regression of the arrested fork DNA, iii) RecF recruits replication proteins to re-establish DNA synthesis following disruption. Surprisingly, the RecF crystal structure reveals a high degree of structural conservation to human Rad50, a double strand break repair protein in humans. The structural similarity between RecF and Rad50 implies a conserved mechanism for initiating repair of DNA ends at replication forks and double strand breaks, respectively. The second aim of this proposal tests this hypothesis by establishing a cellular assay to monitor the repair of chromosomal double strand breaks in E. coli. This approach will provide a system to identify the molecular intermediates associated with the repair of double strand breaks in vivo, a pathway that has proven difficult to characterize in humans and whose details remain poorly understood. PUBLIC HEALTH RELEVANCE: This research is relevant to human health because most genomic instability and mutagenesis results from the replication of damaged DNA. It is widely understood that inaccurate replication in the presence of DNA damage is primary event leading to the formation of cancer and aging in humans. The RecF protein belongs to a ubiquitous group of Recombinational Mediators that include eukaryotic proteins such as Rad52 and BRCA2 which are required to maintain genome stability in the presence of DNA damage. Several human homologs of RecF pathway proteins- BLM, WRN, Rothmund- Thompson syndrome, are associated with genome instability, premature aging, and cancer when mutated. These processes have proven extremely difficult to approach using biochemical or cellular approaches in humans, making E. coli an extremely attractive and appropriate model system for understanding the molecular basis of these complex diseases. Understanding how these genes function is expected to lead to the development of new therapeutics and strategies in the prevention and treatment of cancer and diseases associated with the aging process.